Leptospirosis is considered to be the most widespread, yet neglected, re-emerging zoonotic disease caused by infection with a pathogenic species of the genus Leptospira. Although this disease is prevalent in Bangladesh, the recent epidemiological status has not yet been well documented. In this study, we aimed to determine the prevalence of leptospirosis among febrile patients using different diagnostic methods and to characterize the epidemiological features and species of Leptospira in Mymensingh, north–central Bangladesh. Among the blood samples of 186 patients with suspected leptospirosis who met the inclusion criteria, including having a fever for more than 5 days (November 2021–June 2022), 88 samples (47%) were Leptospira-positive according to IgM LAT, IgM ELISA, or nested PCR (positivity rates: 38%, 37%, and 42%, respectively). Nested PCR showed a significantly higher positivity rate (54%) in patients with a short fever (5–10 day) than the other methods did, with lower rates among those with a longer fever. Leptospirosis cases were more common in males (68%), those 16–45 years of age (70%), residents of rural areas (81%), and farmers (41%). In addition to a fever, myalgia and jaundice were found in more than 70% of the patients, while variable symptoms were observed. The 16S rRNA sequencing analysis revealed that the Leptospira species in all the 22 samples tested were L. wolffii, belonging to the pathogenic subclade P2. This study showed the recent epidemiological features of leptospirosis in Bangladesh, indicating the presumptive predominance of L. wolffii since 2019.